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ISSN : 0250-3360(Print)
ISSN : 2287-5174(Online)
Korean Journal of Breeding Science Vol.44 No.4 pp.450-461
DOI : https://doi.org/10.9787/KJBS.2012.44.4.450

Morphological Characterization of Anther Derived Plants in Minipaprika (Capsicum annuum L.)

Won Hee Kang, Binod Prasad Luitel, Prakash Babu Adhikari, Surendra Lal Shrestha
Department of Horticulture, Kangwon National University
Received on August 9, 2012. Revised on November 13, 2012. Accepted on November 20, 2012

Abstract

This study was done to characterize the anther derived regenerants (R1) including haploids and spontaneous diploidsof minipaprika (cvs.‘Vine sweet-red’ ‘Vine sweet-yellow’ and ‘Vine sweet-orange’) in glasshouse. Eleven haploids (three, sevenand one from red, yellow and orange, respectively) and sixteen spontaneous diploids (five, nine and two from red, yellow andorange, respectively) were grown in plastic pot with three (red, yellow and orange) anther donor (R0) F1minipaprika varieties.Regenerants were characterized for their plant and fruit characters as well as their fruit color and shape. The homozygosity ofspontaneous diploid plants of each population was assessed using simple-sequence repeat (SSR) marker analysis. Haploid plantswere characterized by reduced plant height, small leaves, short petiole and internode and small flower bud and all haploidsshowed the sterility and vice-versa in spontaneous diploid lines. The fruit biometrical traits exhibited greater variation within thespontaneous diploid plants and average value of quantitative traits is lower than standard varieties. MR-4 gave the highest yield(150.5 g) per plant followed by MY-6 (140.0 g) and MY-8 (130.5 g) and the lowest in MY-5 (31.5 g). Morphological markersuch as fruit color further determinedthe microspore origin of androgenic diploids obtained in anther culture of ‘Vine sweet-red’.Of the fifteen spontaneous diploid plants, fourteen plants were identified as doubled haploids using microsatellite markers (SSR),and these homozygous lines are recommended to use in minipaprika breeding program

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INTRODUCTION

In vitro anther culture is the recent breeding method to obtain the haploid and doubled haploid (DH) plants in pepper (Gemesne et al., 2009; Nowaczyk et al., 2009; Pauk et al., 2010; Irikova et al., 2011). The morphological evaluation of doubled haploid lines is important to differentiate the particular lines (Olszewska et al., 2011). The morphological characterization of anther culture derived plants is useful for pepper breeding program (Shrestha et al., 2011). The uniform fruit size with blocky, lamuyo shape, smooth and shiny surface, uniform color, thick and firm pericarp, and good storability are the requirements for developing the new variety in minipaprika. In addition, earliness, fruit quality including flavor and pungency, pedicel length, and multiple fruitedness are important horticultural characters for the pepper breeding (Greenleaf, 1986). 

The in vitro culture of anther follows two modes of androgenesis that lead to the development of the haploids either directly via pollen embryogenesis or indirectly via callus formation (Bajaj, 1983). Since the callus-derived plants show the genetic variation, pepper breeders prefer the plants develop from an unreduced microspore origin. The homozygous dihaploid plantlets derived from microspores have direct value to breeding program and doubled haploids (DHs) have been widely used to develop the varieties in peppers (Pauk et al., 2010; Dolcet-Sanjuan, 2005) and asparagus (Falavignaet al., 1999; Corriolset al., 1990). The ‘spontaneous duplication’ of chromosome during the anther culture results dihaploids in pepper (Sibi et al., 1979; Dumas De Vaulx et al., 1981; Vagera and Havranek, 1985; Vagera, 1990). The pollen-derived homozygous diploid plants show the normal meiotic segregation and they do not loss the desirable characters by segregation. Therefore, it is important to determine the origin of diploid regenerants (Munyon et al., 1989; Olsewska et al., 2011) obtained from anther culture. 

The fruit characters are also very useful for the confirmation on microspore origins of pepper androgenic plants, determined by single particularly recessive genes, e.g. fruit color and shape (Smith, 1950; Pochard, 1977). The origin of anther culture derived regenerants and their homo- and heterozygosity were determined using enzyme markers (Munyon et al., 1989; Dolcet-Sanjuan et al., 1997; Olszewska et al., 2011). Likewise, the confirmation of genetic homogeneity within the plants of particular DH lines and the polymorphism between the different lines is also possible through PCR based DNA markers (Gyulai et al., 2000; Gemesne et al., 2001). The objectives of this study wereto analyze the plant and fruit morphological characters of anther derived regenerants (R1 ) and toconfirm the homozygosity of spontaneous dihaploid plants obtained from the anther culture of minipaprikausing SSR markers. 

MATERIALS AND METHODS

Growing regenerants (R₁) in glasshouse

The number of plants regenerated from anther culture of minipaprika cv. ‘Vine sweet-red’, ‘Vine sweet-yellow’ and ‘Vine sweet-orange’ were 9, 33 and 3, respectively (Luitel, 2012). After the ploidy analysis of regenerated plants, haploids and spontaneously diploid plants were separated from each group and grown at pot (30 cm × 27 cm × 17 cm) in glasshouse, at Kangwon National University (KNU) inFeb. 2012, The haploid plants survived during the acclimatization were 3 (75%), 7 (50.0%) and 1 (100%) in red, yellow, and orange forms respectively, whereas 5 (100%) 9(47.3%), and 2 (100%) diploid plants were survived in red, yellow and orange forms of minipaprika, respectively (Luitel, 2012). The F1 seedlings of red, yellow and orange minipaprika (or anther donor plants, R0) were transplanted in pots for the comparison. The regenerants were named as minipaprika (M) series on the basis of fruit color of their anther donor genotypes. The plants were watered daily and fertilizer was supplied as poly-feed®, soluble nitrogen, phosphorous and potash (NPK) (11-8-34+ 2 Mg) as per the plants requirement.

Morphological characterization of regenerants

Haploids and spontaneous diploids were characterized for their plant height (cm), leaf size (leaf length and width), petiole length, internode length, flower bud size (length and width), nodal color, leaf color and leaf shape after 50 days of transplanting in the pots, and compared with anther donor plants of anther culture. Vernier caliper was used to measure the flower bud size. In diploid plants of each minipaprika, fruit per plant, fruit weight, fruit yield per plant, fruit length and width, fruit shape index (length/ width ratio), fruit volume, pericarp thickness, length of the fruit stalk, fruit stalk weight, fruit set, maturity, fruit size, stalk cavity, fruit lobe number, immature and mature fruit color, fruit shape, fruit shape at apex, fruit surface, and number of seed per fruit were observed. Fruit yield per plant was calculated by counting and weighing all the fruits per plant. Plant and fruit characteristics were evaluated according to the principles given in ‘Descriptors for Capsicum (Capsicum spp.)’ (IPGRI, 1995), and general descriptors developed in minipaprika. 

Identification of homozygosity using SSR markers

Total genomic DNA was extracted from fresh young leaf tissue from anther derived spontaneously diploids (5, 9 and 1 lines from red, yellow, and orange, respectively) and anther donor (parent) plants using a simplified 2% CTAB method (Doyle and Doyle, 1990). 

Seven microsatellite primers; 1.(ACTG)4, 2.(GACA)4, 3.(GATA)4, 4.(GACAC)4, 5.5’-GACAGATAGACAGACA-3’ 6.5’-GACAGATAGACAGATA-3’ and 7. 5’-GACAGACAGATACATA- 3’ were obtained from Macrogen Co. (Seoul, Korea). These primers were initially developed for tomato (Gupta et al., 1994) and later, primers 1 and 2 were used for Capsicum (Gyulai et al., 2000) to identify the doubled haploids. The annealing temperature was 45°C (primers 1 and 2), 43°C (primer 3), 55°C (primer 4), 48°C (primer 5) and 44°C (primers 6 and 7). PCR was performed in 20 μl final volume containing 1.5 μl 10 × buffers, 2.5 mMdNTPs, 25 mM MgCl2 , 0.2 mM of each primer, 1 unit ofTaq DNA polymerase (Taka Ra Ta, Osaka, Japan) and 40 ng of genomic DNA. PCR was carried out in a MyCyclerTM thermocycler (Bio-Rad, California, USA) for 35 cycles and each cycle consisted of the following step; 1 min at 95°C, 1 min at the annealing temperature specified for each primer (above mentioned) and 5 min at 70°C followed by 4°C. Two microliters of the amplified products were mixed with two microliters of loading dye (6X concentrate), then it was loaded in 1% agarose gel, followed by electrophoresis using 1 × TBE buffer (89 mMTris-HCl; pH = 8.3, 89 mM boric acid, 2 mM EDTA) at 55 W for 1 h. The gel was stained with ethidium bromide and photographed under UV light. 

Statistical analyses

The data on plant morphological and fruit characters were taken in anther derived regenerants and donor genotypes of minipaprika. Except plant height, plant and fruit morphological characters were determined at three samples with three readings, and data were analyzed using descriptive statistics (mean and standard deviation) through Microsoft Excel (Version 7). 

RESULTS

Plant morphological characterization

The anther derived plants from ‘Vine sweet-red’ were studied for their morphological characters which are given in Table 1. The average plant height of haploid population was 17.7 cm. Likewise, the average of leaf length and width in haploid plants were 6.8 cm and 3.2 cm, respectively. Short petiole and internode, and small flower bud size were observed in haploid regenerants. In contrast, the average plant height was highest (78.8 cm) with a ranged from 33.0 cm (MR-2) to 115.5 cm (MR-5) in anther derived spontaneous diploid plants. The spontaneous diploid plants of these groups were observed as larger leaf length with width, petiole, internode length, and flower bud size. However, as compared to anther donor plants, the average plant height (78.8 cm), leaf length (9.7 cm), width (5.3 cm), petiole length (3.6 cm), internode length (4.5 cm), flower bud length (4.8 mm), and bud width (3.7 mm) were observed in spontaneous diploids. The nodal pigment was varied green to dark purple in haploids but it was varied from purple to dark purple in diploid plants. Haploids were observed as green leaves but in diploids, it varied from green to dark green leaves. The leaf shape in haploids was varied from ovate to lanceolate whereas it was varied from ovate to deltoid in spontaneous diploids but deltoid leaf shape was observed in F1 cultivar.

Table 1. Plant morphological characters of anther derived plants/regenerants (R1) and donor plants (R0) or F1 hybrid of in minipaprika (Capsicum annuum L. cv. ‘Vine sweet-red’).

The morphological characterization of regenerants obtained from ‘Vine sweet-yellow’ is given in Table 2. In haploids, reduced plant height, leaf size, petiole and internodes length, and flower bud sizes were observed as compared to spontaneous diploids. Within the spontaneous diploids, the plant height ranged from 15.0 cm (MY-3) to 90.0 cm (MY-7) with the average of 44.8 cm. However, the average plant height of spontaneous diploid plants was found three fold smaller than anther donor cultivar. Similarly, as compared to spontaneous diploids, the F1cultivar Vine sweet-yellow exhibited the largest leaf size including length (14.0 cm) and width (7.4 cm), petiole and internode length (6.1 and 6.8 cm) flower bud length (6.4 mm), and width (4.9 mm). The nodal color in haploids was varied from green to purple whereas dark purple pigment was observed in MY-8 but non-pigmented (green) nodal color observed in MY-2. Most of the haploids were observed as green leaves except MY-16 (dark green). Out of nine diploids lines, eight lines were observed as dark green leaves. The leaf shape in haploids was varied from ovate to lanceolate. Except MY-8 (deltoid) and MY-6 (lanceolate), all the diploids observed as ovate leaf but standard cultivars had deltoid leaf shape.

Table 2. Plant morphological characters of anther derived plants/regenerants (R1) and donor plants (R0) or F1 hybrid of minipaprika (Capsicum annuum L. cv. ‘Vine sweet-yellow’).

 The characterization of haploids and diploids obtained from the anther culture of ‘Vine sweet-orange’ are shown in Table 3. The average plant height of diploids was 25.9 cm while the lowest (19.0 cm) plant height was measured in haploid. The spontaneous diploids were also observed as larger leaf size with long petiole and internode, and bigger flower bud size than the haploid. Whereas, the highest plant height(136.0 cm), leaf length (13.1 cm), width (6.8 cm), petiole and internode length (6.6 and 6.7 cm), flower bud length (7.9 mm) and width (4.5 mm) measured in standard cultivar. Likewise, nodal color in diploids ranged from purple to dark purple but green color was marked in the node of haploid. Except F1 cultivar (dark green), all the regenerants were observed as green leaves. The leaves of regenerants were observed as ovate shape except F1 cultivar (deltoid shape).

Table 3. Plant morphological characters of anther derived plants/regenerants (R1) and donor plants (R0) or F1 hybrid of minipaprika (Capsicum annuum L. cv. ‘Vine sweet-orange’).

Fruit characterization

The fruit characters of regenerated diploid plants derived from ‘Vine sweet-red’, ‘Vine sweet-yellow’ and ‘Vine sweetorange’ are given in Table 4. In the regenerants of ‘Vine sweet-red,’ fruit per plant ranged from 3.0 (MR-5) to 8.0 (MR-4) with the average fruit number of 5.0. However, it ranged from 3.0 (MY-2, MY-4, MY-5, and MY-9) to 10.0 (MY-6) in the diploid lines of ‘Vine sweet-yellow’. The average fruit number per plant in spontaneous diploid lines was almost three fold smaller than the standard cultivars (‘Vine sweet-red’, ‘Vine sweet-yellow’ and ‘Vine sweetorange’). Similarly, the fruit weight of diploid population of red minipaprika ranged from 5.6 g (MR-1) to 19.7 (MR-5) with the average of 15.2 g. But in the diploid population of yellow minipaprika, the fruit weight ranged from 10.5 g (MY-5) to 25.0 g (MY-4) with the average of 18.2 g whereas, MO-1 produced 16.0 g fruit. The average fruit weight of standard red, yellow and orange minipaprika varieties were 29.6%, 26.3% and 29.2% higher than the average fruit weight of regenerants in red, yellow and orange, respectively. Fruit yield per plant varied from 35.6 g (MR-1) to 150.5 g (MR-4) with the average of 69.2 g in the diploid lines of red minipaprika, whereas, it ranged from 31.5 g (MY-5) to 140.0 g (MY-6) with the average of 83.8 g in the population of yellow minipaprika. The average yield obtained from the population of red, yellow and orange were 77.1%, 78.4%, and 77.6% lowered as compared to the standard varieties of red, yellow and orange, respectively. 

Table 4. Fruit yield and quality characters in anther derived diploid lines/regenerants (R1) and donor plants (R0) or F1 hybrid ofminipaprika(Capsicum annuumcv. ‘Vine sweet’).

With respect to fruit size, it was also varied among the diploid population of each minipaprika. In the regenerants of ‘Vine sweet-red’, fruit length varied from 35.7 mm (MR-1) to 64.6 mm (MR-3) with the average of 50.2 mm. Likewise, in the regenerants of ‘Vine sweet-yellow’, the fruit length varied from 34.8 mm (MY-5) to 66.7 (MY-7) with an average fruit length of 53.4 mm. However, average fruit length was higher in standard minipaprika red (54.8 mm), yellow (57.6 mm) and orange (55.3 mm) varieties. The average fruit width was 26.0 mm with a range from 19.5 mm (MR-1) to 36.9 (MR-5) in the regenerants of red minipaprika. In the regenerants of yellow minipaprika, the lowest (22.4 mm) fruit width was measured in MY-5 and the highest was (37.2 mm) in MY-4 followed by MY-3 (35.6 mm) with the average of 27.9 mm fruit width. The fruit width measured was 29.2 mm in MO-1. In contrast, the average fruit width of original cultivars was higher as compared to the regenerants of each population. Variation in fruit shape index was observed in the anther derived plant population and standard cultivars. The highest fruit shape index was measured in MY-7 followed by MR-3 (Fig. 1). 

Fig. 1. Fruit shape index (FSI) variation in anther derived diploids and donor genotypes of minipaprika (Capsicum annuum L.). Vertical bar indicates ± standard deviation(n = 3).

In the regenerants of ‘Vine sweet-red’, the fruit volume was averaged 11.4 cc which ranged from 8.2 cc (MR-1) to 14.9 cc (MR-5). Likewise, the highest (20.0 cc) fruit volume was recorded in MY-4 and the lowest (6.7 cc) in MY-5 with the average of 11.5 cc fruit volume. The fruit volume produced in original cultivars was highest than the regenerants of each minipaprika. The average pericarp thickness measured was 4.3 mm with a range from 4.1 mm to 4.8 mm in the regenerants of ‘Vine sweet-red’. However, it was varied from 3.1 mm (MY-2) to 5.5 mm (MY-3) with the average of 3.9 mm in regenerants of ‘Vine sweetyellow’ minipaprika. Likewise, fruit stalk length (pedicel length) ranged from 14.1 mm (MR-2) to 28.2 (MR-4) with the average of 19.7 mm in the regenerants of red minipaprika. Likewise, the fruit stalk length averaged was 18.4 mm with a range from 13.9 mm (MY-9) to 26.3 mm (MY-4) in the regenerants of yellow minipaprika. MO-1 was measured to have 15.5 mm fruit stalk length. The average fruit stalk length was also higher in the standard varieties than the anther derived regenerants of each minipaprika. Similarly, fruit stalk’s weight varied among the regenerants of all forms of minipaprika but the average fruit stalk weight was also higher in the standard cultivars. 

Variation of fruit morphological characters and seed number in the anther regenerated diploid plants are shown in Table 5. In the regenerants of ‘Vine sweet-red’, and ‘Vine sweet-Yellow’, MR-1, MR-4, MY-1, MY-3 and MY-6 was recorded as high fruit setting lines, whereas, MR-3, MY-7 and MY-8 categorized into medium fruit setting lines, and remaining lines were categorized into low fruit setting diploid lines. As compared to standard varieties, MR-3, MR-4, MY-3, MY-6 and MO-1 were identified as early maturing varieties and remaining lines were categorized into medium maturing type. The fruits obtained from MR-1 and MY-5 lines were identified as of small size, whereas MR-2, MR-3, MR-4, MY-1, MY-2, MY-6, MY-7, MY-9, and MO-1 and bore medium fruit size, and MR-5, MY-3, MY-4, and MY-8 produced bigger sized fruit. The shallow stalk cavity was identified in the fruits of MR-2 and MY-7, whereas MR-1, MR-4, MY-1, MY-3 and MY-6 had deep stalk cavity, MR-5 had no cavity in fruits, and the fruits of remaining lines were identified as medium stalk cavity. Fruit lobe ranged from 2.0 to 4.0. MR-5 and MY-3 produced four lobed fruits, MY-4 bore 3 lobed fruit, and remaining lines were characterized as two lobed fruits. The dark green fruit color was observed in immature fruit stage of MR-4, MY-2, MY-3, MY-6 and MY-7 and remaining lines were observed as green colored at immature stage. The color variation in mature fruit ranged from red to yellow in regenerated plants of ‘Vine sweet-red’. MR-1, MR-2, and MR-5 bore the yellow color fruit in the regenerants of ‘Vine sweet-red’, while the fruits of MR-3 and MR-4 had characterized as light red and dark red color, respectively. In the population of ‘Vine sweet-yellow’, MY-1, MY-2, MY-3, MY-4, MY-5, MY-6, and MY-9 bore the yellow colored fruits, whereas fruits of MY-7 and MY-8 were observed to be of red and orange color, respectively. MY-7 had elongated fruit shape, whereas MR-5 and MY-3 were observed to have blocky shaped fruit and triangular or glamour fruit shape was observed in the remaining lines. The fruit morphology of anther derived diploid regenerants of ‘Vine sweet-red, yellow and orange forms is shown in Fig. 2 and Fig. 3, respectively. All the diploid lines were observed to have smooth skin fruit surface except MR-2 and MY-5 (semi-wrinkled), and MY-5 had russet skin. The seed number per fruit varied from 6.0 (MR-1) to 37.3 (MR-4) in the regenerated plants in ‘Vine sweet-red’. In the population of ‘Vine sweet-yellow’, the seed number per fruit varied from 5.3 (MY-7) to 28.0 (MY-5) and seed number was 10.8 in MO-1. The average seed numbers per fruit in spontaneous diploids of each minipaprika were also smaller than the standard varieties that of ‘Vine sweet-red’, ‘Vine sweet-yellow’ and ‘Vine sweet-orange’. 

Table 5. Fruit morphological characters in anther derived diploid plants/regenerants (R1) and donor plants (R0) or F1 hybrid ofminipaprika(Capsicum annuumcv. ‘Vine sweet’)

Fig. 2. Fruit morphology of anther donor diploid plants of minipaprika cv. ‘Vine sweet-red’ and anther derived spontaneous doubled haploids. A, F1 fruit, B-E, five lines obtained from ‘Vine sweet-red’ F1 anther culture, A, Anther donor F1 hybrid ‘Vine sweet-red’ B, C, D, E and F indicate the fruits of MR-1, MR-2, MR-3, MR-4, and MR-5, respectively.

Fig. 3. Fruit morphology of anther donor diploid plants and anther derived spontaneous doubled haploids in minipaprika. A, F1 fruit (‘Vine sweet-yellow’), and nine lines B, C, D, E, F, G, H, I, and J indicate the fruits of spontaneous doubled haploid MY-1, MY-2, MY-3, MY-4, MY-5, MY-6, MY-7, MY-8, and MY-9 derived from ‘Vine sweet-yellow’ respectively. H indicates the spontaneous diploid line MO-1 derived from ‘Vine sweet-orange’.

SSR marker analysis

Seven microsatellites primers were screened. Out of seven primers, three (ACTG)4, (GACA)4 and (GAGAC)4 primers could distinguish different lines. In red minipaprika cultivar ‘Vine sweet-red’, the mother plant showed five amplification bands while those plants originated from the anther culture of ‘Vine sweet-red’ exhibited the two amplified band except MR-4 at the same level demonstrating that they were doubled haploids (Fig. 4A). MR-4 showed almost similar pattern with mother plant indicating that this line might be normal diploid having a somatic origin. In yellow minipaprikacultivar ‘Vine sweet-yellow’, SSR marker (GACA)4 amplified four bands in mother plant. In contrast, the regenerated plants exhibited three amplified bands indicating that they were doubled haploids (Fig. 4B). A similar observation was also made in orange minipaprika population (Fig. 4C). 

Fig. 4. An ethidium bromide stained agarose gel electrophoresis showing DNA patterns. A-C, indicate the amplified bands by primer (ACTG)4, (GACA)4 and (GAGAC)4 from the DNA of five, nine and one diploid lines derived from anther culture of ‘Vine sweet- red’, Vine sweet- yellow’(* indicates the presence of band) and ‘Vine sweet-orange’, respectively. Lane M, 1-kb ladder, as a DNA size standard. Lane VS-R, VS-Y and VS-O indicate as ‘Vine sweet-red’, ‘Vine sweet-yellow’ and ‘Vine sweet-orange’, respectively.

DISCUSSION

In this study, eleven haploid and sixteen diploid plants were evaluated for their plant morphological characters. We found the variability within the haploid plants in their plant characters. Overall, haploids population in each population had recorded lower plant height, narrower leaf size, shorter petiole and internode length as well as smaller flower bud length as compared to spontaneous diploid plants. We observed the haploids that underwent into the flowering stage but none of the haploids were found to bear the fruits. In the absence of homologous chromosomes, the meiosis is abnormal and as a result, no viable gametes are formed and this is the reason of haploid being sterile. Supena et al. (2006) reported the smaller and narrow leaves in haploids than in diploid plants regenerated from microspore derived embryos of the hot pepper genotypes. Shrestha et al. (2010, 2011) reported shorter plant height, smaller leaf and flower bud in the haploids of sweet pepper cvs. ‘Special’ and ‘Boogie’, respectively. On the other hand, variation in plant and fruit characters was observed in anther derived spontaneous diploid population. Diploid plants derived from minipaprika-red showed more variation in plant height than the plants of minipaprikayellow and orange. Likewise, variation in leaf size, petiole and internode length, flower bud size, nodal color, leaf color, and leaf shape was observed within the diploid lines of each minipaprika. We also observed the variability in fruit characters among the spontaneous diploid lines of each minipaprika. Such variation in quantitative traits had an additive character. However, the mean values of plant and fruit characters in androgenic diploid plants of each group were lower than the standard varieties. Vagera (1990) reported the similar results in pepper. Paepe et al. (1981) found the difference in morphological characters between double haploid plants and original cultivar in Nicotinasylvestris. Some authors (Oinuma and Yoschida 1975; Arcia et al., 1978) have observed double haploid lines that differ from their parent plants. The double haploid lines or pure lines have narrow genetic base (Acquaah, 2007) and this might attributed to less vigorousness, reduced plant height, low fruit setting, lower fruit yield with fewer seed number in spontaneous double haploids than the hybrid cultivars. Brown and Wernsman (1982) had mentioned that genetic differences between diploids lines and their source cultivar are largely due to their nuclear origin. Likewise, Shrestha et al. (2011) observed the interline variation in spontaneous diploid lines of bell pepper cultivar cv. ‘Boogie’. The interline variability comes from a genetic diversity of microspores which is an effect of random gene segregation during meiosis (Gemesne et al., 2001) and our results agree with this finding. This difference exist within the diploid lines might be due to the result of naturally produced variation. Besides, most of the agronomic variation could orginate from the changes that occur in the DNA of the vegetative pollen grain cell. 

Fruit yield, fruit size, pericarp thickness, pedicel length and fruit shape are quantitative traits which are governed by polygenes, and also influenced by the environment. However, these are important plant characters that should not be omitted in the process of DHs evaluation. On the other hand, fruit color is a qualitative trait, not influenced by the environment, can be applied to mark the origin of androgenic pepper plants. The inheritance of mature fruit color is controlled by three independent genes Y, C1 and C2 (Hurtado-Hernandez &Smith, 1985). The segregation of these genes produces the range of colors from white to red in mature fruit. A dominant allele at Y is required for the production of capsorubin and capsanthin, the carotenoids that determine red color. The recessive allele at this locus determines yellow or orange fruit. The minipaprika cv. ‘Vine sweet-red’ is F1hybridand red fruit color is determined by y+y+, cl+cl+. We obtained the three double haploid lines with yellow fruit, and two double haploid lines with light red and dark red color from the anther culture of minipaprikared genotype. This must have formed as a result of spontaneous diploidization of haploid embryos, which developed from the microspore with recessive allele y indicating that these spontaneous diploids are fully homozygous lines resulting three lines in yellow colored fruit. Our result confirms the finding of Olszewska et al. (2011) and they had also found the anther culture derived diploids with yellow fruit from the red sweet pepper variety. Shrestha et al. (2010) had obtained the yellow fruit in spontaneous diploid line from the anther culture of red sweet pepper cv. ‘Debora’. Additionally, we found red and orange fruits in the doubled haploid lines derived from the yellow minipaprika. The genetic change during spontaneous doubling of chromosome might be the reason for the development of such trait. Shreshta et al. (2011) had also obtained red fruit color of androgenic lines from the anther culture of sweet pepper cv. ‘Boogie’ (orange), ‘Bossanova’ (yellow) and ‘Fiesta’ (yellow). Pochard (1977) had mentioned that round fruit is determined by dominant allele (o+) and (o+/o) determines the round fruit whereas recessive allele at this locus results the elongated fruit shape. But in this study, we did not observe round fruit in the double haploids of each minipaprika. However, we observed the blocky shape fruit in two doubled haploid lines developed from triangular or glamour shaped yellow minipaprika genotype. Doubled haploid exhibited the variation primarily due to additive gene effect. Besides, we analyzed the spontaneous diploid plants using SSR markers and fourteen plants were identified as the homozygous lines. The lines differed genetically from the anther donor plant, and therefore, they were not of somatic cell origin except one diploid. In addition to SSR markers, RAPD and RFLP are widely used molecular markers in Capsicum and these markers can be an important tool for the confirmation of homozygosity. 

In conclusion, this study analyzed the plant morphological characters of regenerants including haploids and spontaneous diploids. Additionally, this study examined the homozygosity of spontaneous diploid regenerants obtained from each minipaprika genotype. The plant and fruit characterization of doubled haploids helped to identify the particular homozygous lines. The spontaneous doubled haploids exhibited more variation in fruit yield. This androgenic variation observed within the doubled haploid is under the nuclear control. Overall, the interline variability within the doubled haploid lines is very important for breeding new ideotype. The plant morphological marker trait such as fruit color analysis of regenerants helped to identify the spontaneously doubled haploids derived from the ‘Vine-sweet red’. Besides, the SSR marker analysis confirmed the homozygosity of anther derived spontaneous diploid lines as doubled haploids. These doubled haploids are recommended for further development of inbred lines and heterosis breeding in minipaprika. 

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